Microbiome analysis of healthy and diseased sponges Lubomirskia baicalensis by using cell cultures of primmorphs
- Published
- Accepted
- Subject Areas
- Bioinformatics, Cell Biology, Microbiology, Molecular Biology, Freshwater Biology
- Keywords
- Lubomirskia baikcalensis, Primmorphs, Symbionts, Opportunistic pathogen
- Copyright
- © 2019 Chernogor et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2019. Microbiome analysis of healthy and diseased sponges Lubomirskia baicalensis by using cell cultures of primmorphs. PeerJ Preprints 7:e27851v1 https://doi.org/10.7287/peerj.preprints.27851v1
Abstract
Background. Freshwater sponges (Demosponges, Lubomirskiidae) are dominated in the littoral zone Lake Baikal in the biomass of benthic organisms and represent complex consortia of many species of eukaryotes and prokaryotes. A distinctive feature of sponges from Lake Baikal is their ability to live in symbiosis with various kinds of chlorophyll containing microalgae. Recently there have been massive diseases and the death of freshwater sponges. The etiology and ecology of these events remain unknown. Purpose. The purpose of the research was to use cell culture of primmorphs in vitro to study the microbiomes of healthy and diseased sponges to show the transmission of pathogenic agents from diseased sponges to cell cultures. Methods. The cell culture of primmorphs sponge Lubomirskia baicalensis was used to study microbiome communities in diseased and sick sponges in comparison with healthy sponge with subsequent sequencing of gene 16S rRNA and analysis of changes in microbiomes. Results. Results this study were show that use of cell culture of primmorphs in vitro is equivale of healthy sponge. Microbial community of healthy sponge and primmorphs was grouped separately from the community of diseased sponges and infected primmorphs, which confirms the suitability the cell culture of primmorphs, as a model sponge system. We found the mass death of green symbionts (Chlorophyta) and a shift in the microbial communities of sponges/primmorphs, associated with increase in relative abundant of different phyla Bacteroidetes and Proteobacteria with dominated families Flavobacteriaceae and Burkholderiaceae, Moraxellaceae in diseased sponges and infected cell cultures of primmorphs. Conclusions. This approach allowed us, using the cell culture of primmorphs, to identify potential opportunistic bacteria that can work together, which possibly enhances their action. The primmorphs system described here is a powerful new model system for studying basic mechanisms of the development of sponge disease, which will be valuable in future studies.
Author Comment
The manuscript presented is of a methodical character and is devoted to the description the disease of the Baikal sponges using the model cell culture of primmorphs. We showed in our research that the model works and this study need in further continuation to find out the causes of the death of Baikal sponges.
Supplemental Information
Samples of the healthy sponge and primmorphs
(A) The healthy freshwater Baikal sponge L. baicalensis, (B) cell culture of primmorphs of L. baicalensis obtained from the sponge. Scale bars are 5 mm. Canon EOS 200D digital camera.
Samples of diseased sponges
(A) The diseased freshwater Baikal sponge L. baicalensis. (B) The sick freshwater Baikal sponge L. baicalensis. Canon EOS 200D digital camera.
Experimental design study of the microbiomes of healthy, diseased sponges and primmorphs
Notes. 1 The suspensions of microorganisms from diseased sponge. 2 The suspensions of microorganisms from sick sponge.
Light and fluorescence images cell cultures of primmorphs of sponge L. baicalensis.
(A) Light microscopy, showing green microalgae located within amoebocytes in the healthy cell culture of primmorphs. Arrows show sponge amoebocytes within microalgae (B) Fluorescence microscopy, showing red autofluorescence of chlorophyll-containing intracellular of green algae in the healthy primmorphs. (C) The primmorphs infected with cellular suspension from the diseased sponge observed the death of green algae symbionts, sponge cell (indicated by arrow). Shown massive numbers, of different bacteria at 7 day (indicated by arrow). (D) Fluorescence microscopy, showing the death of microalgae (red color) in infected primmorphs from diseased sponge at 7 day. Bacteria shown blue color. (E) The primmorphs infected with cellular suspension from the diseased sponge for 21 days, shown residues of green algae in cell culture of primmorphs and huge biomass of bacteria (indicated by arrow). (F) Fluorescence microscopy showing death of green algae primmorphs infected suspension from the diseased sponge and massive of different bacteria for 21 day. Bacteria in infected primmorphs, shown blue color. Samples of primmorphs stained with the NucBlue Live ReadyProbes reagent for fluorescence microscopy. Scale bars: 10 μm.
SEM images of cell cultures of primmorphs
(A) The epithelial surface of healthy cultures were clean, flat and smooth. (B) The surface of the primmorphs infected with the cellular suspension from the diseased sponge. Observed melting of the epithelial cells of sponge, increased different bacteria at 7 day. (C) The primmorphs infected with cellular suspension from the diseased sponge, the death of green algae symbionts, sponge cells and massive growth of different bacteria for 21 day. (D) Bio-cake formed in infected cultures of primmorphs from diseased sponge at 30 day. Scale bars are 1 μm.
The alpha-diversity indexes ( Chao1 and Shannon index ) of the data distribution
A) The distribution between the group’s adult sponges and primmorphs. B) The distribution between the groups of healthy and diseased of sponges and primmorphs. Samples were referred to Table 1. We did not find significant differences between diversity the adult sponges and primmorphs by using Shannon index. The alpha-diversity indices (Shannon index) have significant difference between healthy and diseased groups (Table 3, Fig. 6 B).
The beta-diversity results of PCoA indicating the data distribution between groups
Samples of the healthy sponge and primmorphs are grouped into one cluster and differ significantly from the group of diseased. Samples were referred to Table 1.
Taxonomic profiles of the microbial communities at the phylum level
Relative abundance of reads assigned to phyla (to %). Samples were referred to Table 1.
Heatmap showing the family with significant differences of relative abundances amongst the two groups
Sample ID are referred to Table 1. Heatmap based on the scale of 0-8 log.
Samples of sponges L. baicalensis and cell culture of primmorphs
Samples were collected from the strait Olkhon Vorota, Lake Baikal.
Summary of microbial communities in sponges and primmorphs
Notes. The name of samples: SH (Healthy sponge); PH1 (Primmorphs for 1 day); PH14 (Primmorphs for 14 day); SD (Bleached sponge); PD (Primmorphs diseased); PID (Primmorphs infected by diseased sponge ); SS (Sick sponge); PIS (Primmorphs infected by sick sponge). Samples IDs are referred to Table 1.
The alpha-diversity indices (Сhao1, Shannon index)
The alpha-diversity were calculated using the QIIME2 software to establish the abundance and diversity of the sequences. Notes. Samples IDs are referred to Table 1.
Results pairwaise PERMANOVA test
Notes. Indicates p-value < 0.05.
Abundance and significant difference between the two groups at the phylum/family level
Notes. (to %). Colored lines indicate a shift in microbial communities in healthy and diseased groups.